Cobblestone appearance with large dark nuclei; during proliferation, cells are small and evenly sized, display a high mitotic index and show no presence of smooth muscle cells.

Growth Properties

Adherent

Biosafety Level

1

[These primary cells are not known to harbor an agent recognized to cause disease in healthy adult humans. Handle as a potentially biohazardous material under at least Biosafety Level 1 containment. Cells derived from primate lymphoid tissue may fall under the regulations of 29 CFR 1910.1030 Bloodborne Pathogens.

Appropriate safety procedures should always be used with this material. Laboratory safety is discussed in the following publication: Biosafety inMicrobiological and Biomedical Laboratories, 5th ed. HHS Publication No. (CDC) 93-8395. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Washington DC: U.S. Government Printing Office; 2007. The entire text is available online at http://www.cdc.gov/biosafety/publications/bmbl5/index.htm.]

Human Material Precaution

All tissues used for isolation are obtained under informed consent and conform to HIPAA standards to protect the privacy of the donor’s personal health information. It is best to use caution when handling any human cells. We recommend that all human cells be accorded the same level of biosafety consideration as cells known to carry HIV. With infectious virus assays or viral antigen assays, even a negative test result may leave open the possible existence of a latent viral genome.

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

The HPAEC cells are cryopreserved in the second passage to ensure the highest purity, coupled with high viability and plating efficiency.

A complete solution to propagate pulmonary artery endothelial cells in low serum conditions with or without the addition of human recombinant VEGF. Use of the Endothelial Cell Growth Kit-VEGF will support a faster rate of proliferation because of the presence of several purified recombinant human (rh) growth factors (rh VEGF, rh EGF, rh FGF basic and rh IGF-1) combined with heparin and hydrocortisone. Use of the Endothelial Cell Growth Kit-BBE, which contains Bovine Brain Extract (BBE), is recommended if a more "authentic," less defined culture medium is desired.

Complete Growth Medium

Obtain one growth kit from the freezer; make sure that the caps of all components are tight.

Thaw the components of the growth kit just prior to adding them to the basal medium. It is necessary to warm the L-glutamine component in a 37°C water bath and shake to dissolve any precipitates prior to adding to the basal medium.

Decontaminate the external surfaces of all growth kit component vials and the basal medium bottle by spraying them with 70% ethanol.

Using aseptic technique, and working in a laminar flow hood or biosafety cabinet, transfer the volume of each growth kit component, as indicated in either Table 1 or 2, to the bottle of basal medium using a separate sterile pipette for each transfer.

Table 1. If using the Endothelial Cell Growth Kit-BBE (ATCC® PCS-100-040), add the indicated volume for each component:

Component

Volume

Final Concentration

Bovine Brain Extract (BBE)

1.0 mL

0.2%

rh EGF

0.5 mL

5 ng/mL

L-glutamine

25.0 mL

10 mM

Heparin sulfate

0.5 mL

0.75 Units/mL

Hydrocortisone hemisuccinate

0.5 mL

1 µg/mL

Fetal Bovine Serum

10.0 mL

2%

Ascorbic acid

0.5 mL

50 µg/mL

Table 2. If using the Endothelial Cell Growth Kit-VEGF (ATCC® PCS-100-041), add the indicated volume for each component:

Component

Volume

Final Concentration

rh VEGF

0.5 mL

5 ng/mL

rh EGF

0.5 mL

5 ng/mL

rh FGF basic

0.5 mL

5 ng/mL

rh IGF-1

0.5 mL

15 ng/mL

L-glutamine

25.0 mL

10 mM

Heparin sulfate

0.5 mL

0.75 Units/mL

Hydrocortisone hemisuccinate

0.5 mL

1 µg/mL

Fetal Bovine Serum

10.0 mL

2%

Ascorbic acid

0.5 mL

50 µg/mL

Antimicrobials and phenol red are not required for proliferation but may be added if desired. The recommended volume of either of the optional components (GA solution or PSA solution) to be added to the complete growth media is summarized in Table 3.

Warm both the Trypsin-EDTA for Primary Cells (ATCC PCS-999-003) and the Trypsin Neutralizing Solution (ATCC PCS-999-004) to room temperature prior to dissociation. Warm complete growth medium to 37°C prior to use with the cells.

For each flask, carefully aspirate the spent media without disturbing the monolayer.

Rinse the cell layer two times with 3 to 5 mL D-PBS (ATCC 30-2200) to remove residual traces of serum.

Add pre-warmed trypsin-EDTA solution (1 to 2 mL for every 25 cm2) to each flask.

Gently rock each flask to ensure complete coverage of the trypsin-EDTA solution over the cells, and then aspirate the excess fluid off of the monolayer.

Observe the cells under the microscope. When the cells pull away from each other and round up (typically within 3 to 5 minutes), remove the flask from the microscope and gently tap it from several sides to promote detachment of the cells from the flask surface.

When the majority of cells appear to have detached, quickly add an equal volume of Trypsin Neutralizing Solution (ATCC PCS-999-004) to each flask. Gently pipette or swirl the culture to ensure all of the trypsin-EDTA solution has been neutralized.

Transfer the dissociated cells to a sterile centrifuge tube and set aside while processing any remaining cells in the flask.

Add 3 to 5 mL D-PBS (ATCC 30-2200) to the flask to collect any additional cells that might have been left behind.

Transfer the cell/D-PBS suspension to the centrifuge tube containing the trypsin-EDTA-dissociated cells.

Repeat steps 10 and 11 as needed until all cells have been collected from the flask.

13. Centrifuge the cells at 150 x g for 3 to 5 minutes.

Aspirate the neutralized dissociation solution from the cell pellet and resuspend the cells in 2 to 8 mL fresh, pre-warmed, complete growth medium.

Count the cells and seed new flasks at a density between 2,500 and 5,000 cells per cm2.
16. Place newly seeded flasks in a 37°C, 5% CO2, incubator for at least 24 to 48 hours before processing the cells further. Refer to Maintenance for guidelines on feeding.

Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.